Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multi-subunit protein complexes, this method has proven challenging due to technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis.
Proteins form stable and dynamic multi-subunit complexes under different physiological conditions to maintain cell viability and normal cell homeostasis. Detailed knowledge of protein interactions and protein complex structures is fundamental to understanding how individual proteins function within a complex and how the complex functions as a whole. However, structural elucidation of large multi-subunit protein complexes has been difficult due to lack of technologies which can effectively handle their dynamic and heterogeneous nature. Traditional methods such as nuclear magnetic resonance (NMR) analysis and X-ray crystallography can yield detailed information on protein structures; however, NMR spectroscopy requires large quantities of pure protein in a specific solvent while X-ray crystallography is often limited by the crystallization process.
In recent years, chemical cross-linking coupled with mass spectrometry (MS) has become a powerful method for studying protein interactions. See for example the disclosures of Sinz, A. (2003) Chemical Cross-Linking and Mass Spectrometry for Mapping Three-Dimensional Structures of Proteins and Protein Complexes. J Mass Spectrom. 38, 1225-1237; Sinz, A. (2006) Chemical Cross-Linking and Mass Spectrometry to Map Three-Dimensional Protein Structures and Protein-Protein Interactions. Mass Spectrom Rev 25, 663-682; and Leitner, A., Walzthoeni, T., Kahraman, A., Herzog, F., Rinner, O., Beck, M., and Aebersold, R. (2010) Probing Native Protein Structures by Chemical Cross-Linking, Mass Spectrometry and Bioinformatics. Molecular & Cellular Proteomics 9, 1634-1649. Chemical cross-linking stabilizes protein interactions through the formation of covalent bonds and allows the detection of stable, weak and/or transient protein-protein interactions in native cells or tissues See for example the disclosures of Sinz, A. (2010) Investigation of Protein-Protein Interactions in Living Cells by Chemical Crosslinking and Mass Spectrometry. Anal Bioanal Chem 397, 3433-3440; Vasilescu, J., Guo, X., and Kast, J. (2004) Identification of Protein-Protein Interactions Using in Vivo Cross-Linking and Mass Spectrometry. Proteomics 4, 3845-3854; Guerrero, C., Tagwerker, C., Kaiser, P., and Huang, L. (2006) An Integrated Mass Spectrometry-Based Proteomic Approach: Quantitative Analysis of Tandem Affinity-Purified in Vivo Cross-Linked Protein Complexes (Qtax) to Decipher the 26 S Proteasome-Interacting Network. Mol Cell Proteomics 5, 366-378; Tagwerker, C., Flick, K., Cui, M., Guerrero, C., Dou, Y., Auer, B., Baldi, P., Huang, L., and Kaiser, P. (2006) A Tandem Affinity Tag for Two-Step Purification under Fully Denaturing Conditions: Application in Ubiquitin Profiling and Protein Complex Identification Combined with in Vivocross-Linking. Mol Cell Proteomics 5, 737-748; Guerrero, C., Milenkovic, T., Przulj, N., Kaiser, P., and Huang, L. (2008) Characterization of the Proteasome Interaction Network Using a Qtax-Based Tag-Team Strategy and Protein Interaction Network Analysis. Proc Natl Acad Sci U.S.A 105, 13333-13338; and Kaake, R. M., Milenkovic, T., Przulj, N., Kaiser, P., and Huang, L. (2010) Characterization of Cell Cycle Specific Protein Interaction Networks of the Yeast 26s Proteasome Complex by the Qtax Strategy. J Proteome Res 9, 2016-2019. In addition to capturing protein interacting partners, many studies have shown that chemical cross-linking can yield low-resolution structural information about the constraints within a molecule. See for example the disclosures of Sinz, A. (2006) Chemical Cross-Linking and Mass Spectrometry to Map Three-Dimensional Protein Structures and Protein-Protein Interactions. Mass Spectrom Rev 25, 663-682; Leitner, A., Walzthoeni, T., Kahraman, A., Herzog, F., Rinner, O., Beck, M., and Aebersold, R. (2010) Probing Native Protein Structures by Chemical Cross-Linking, Mass Spectrometry and Bioinformatics. Molecular & Cellular Proteomics 9, 1634-1649; and Back, J. W., de Jong, L., Muijsers, A. O., and de Koster, C. G. (2003) Chemical Cross-Linking and Mass Spectrometry for Protein Structural Modeling. J Mol Biol. 331, 303-313, or protein complex, as disclosed in Rappsilber, J., Siniossoglou, S., Hurt, E. C., and Mann, M. (2000) A Generic Strategy to Analyze the Spatial Organization of Multi-Protein Complexes by Cross-Linking and Mass Spectrometry. Anal Chem. 72, 267-275; Maiolica, A., Cittaro, D., Borsotti, D., Sennels, L., Ciferri, C., Tarricone, C., Musacchio, A., and Rappsilber, J. (2007) Structural Analysis of Multiprotein Complexes by Cross-Linking, Mass Spectrometry, and Database Searching. Mol Cell Proteomics 6, 2200-2211; and Chen, Z. A., Jawhari, A., Fischer, L., Buchen, C., Tahir, S., Kamenski, T., Rasmussen, M., Lariviere, L., Bukowski-Wills, J. C., Nilges, M., Cramer, P., and Rappsilber, J. (2010) Architecture of the Rna Polymerase Ii-Tfiif Complex Revealed by Cross-Linking and Mass Spectrometry. Embo J 29, 717-726. The application of chemical cross-linking, enzymatic digestion, and subsequent mass spectrometric and computational analysis for the elucidation of three dimensional protein structures offers distinct advantages over traditional methods due to its speed, sensitivity, and versatility. Identification of cross-linked peptides provides distance constraints that aid in constructing the structural topology of proteins and/or protein complexes. Although this approach has been successful, effective detection and accurate identification of cross-linked peptides as well as unambiguous assignment of cross-linked sites remain extremely challenging due to their low abundance and complicated fragmentation behavior in MS analysis. See for the example the disclosures of Sinz, A. (2006) Chemical Cross-Linking and Mass Spectrometry to Map Three-Dimensional Protein Structures and Protein-Protein Interactions. Mass Spectrom Rev 25, 663-682; Leitner, A., Walzthoeni, T., Kahraman, A., Herzog, F., Rinner, O., Beck, M., and Aebersold, R. (2010) Probing Native Protein Structures by Chemical Cross-Linking, Mass Spectrometry and Bioinformatics. Molecular & Cellular Proteomics 9, 1634-1649; Back, J. W., de Jong, L., Muijsers, A. O., and de Koster, C. G. (2003) Chemical Cross-Linking and Mass Spectrometry for Protein Structural Modeling. J Mol Biol. 331, 303-313; and Schilling, B., Row, R. H., Gibson, B. W., Guo, X., and Young, M. M. (2003) Ms2assign, Automated Assignment and Nomenclature of Tandem Mass Spectra of Chemically Crosslinked Peptides. J Am Soc Mass Spectrom. 14, 834-850. Therefore, new reagents and methods are urgently needed to allow unambiguous identification of cross-linked products and to improve the speed and accuracy of data analysis to facilitate its application in structural elucidation of large protein complexes.
A number of approaches have been developed to facilitate MS detection of low abundance cross-linked peptides from complex mixtures. These include selective enrichment using affinity purification with biotinylated cross-linkers, for example, as described in Trester-Zedlitz, M., Kamada, K., Burley, S. K., Fenyo, D., Chait, B. T., and Muir, T. W. (2003) A Modular Cross-Linking Approach for Exploring Protein Interactions. J Am Chem Soc. 125, 2416-2425; Tang, X., Munske, G. R., Siems, W. F., and Bruce, J. E. (2005) Mass Spectrometry Identifiable Cross-Linking Strategy for Studying Protein-Protein Interactions. Anal Chem 77, 311-318; and Chu, F., Mahrus, S., Craik, C. S., and Burlingame, A. L. (2006) Isotope-Coded and Affinity-Tagged Cross-Linking (Icatxl): An Efficient Strategy to Probe Protein Interaction Surfaces. J Am Chem Soc 128, 10362-10363, and click chemistry with alkyne-tagged (Chowdhury, S. M., Du, X., Tolic, N., Wu, S., Moore, R. J., Mayer, M. U., Smith, R. D., and Adkins, J. N. (2009) Identification of Cross-Linked Peptides after Click-Based Enrichment Using Sequential Collision-Induced Dissociation and Electron Transfer Dissociation Tandem Mass Spectrometry. Anal Chem 81, 5524-5532) or azide tagged cross-linkers, see for example Kasper, P. T., Back, J. W., Vitale, M., Hartog, A. F., Roseboom, W., de Koning, L. J., van Maarseveen, J. H., Muijsers, A. O., de Koster, C. G., and de Jong, L. (2007) An Aptly Positioned Azido Group in the Spacer of a Protein Cross-Linker for Facile Mapping of Lysines in Close Proximity. Chembiochem 8, 1281-1292; and Nessen, M. A., Kramer, G., Back, J., Baskin, J. M., Smeenk, L. E., de Koning, L. J., van Maarseveen, J. H., de Jong, L., Bertozzi, C. R., Hiemstra, H., and de Koster, C. G. (2009) Selective Enrichment of Azide-Containing Peptides from Complex Mixtures. J Proteome Res 8, 3702-3711. In addition, Staudinger ligation has recently been shown to be effective for selective enrichment of azide-tagged cross-linked peptides (Vellucci, D., Kao, A., Kaake, R. M., Rychnovsky, S. D., and Huang, L. (2010) Selective Enrichment and Identification of Azide-Tagged Cross-Linked Peptides Using Chemical Ligation and Mass Spectrometry. J Am Soc Mass Spectrom 21, 1432-1445). Apart from enrichment, detection of cross-linked peptides can be achieved by isotope-labeled, as described in Collins, C. J., Schilling, B., Young, M., Dollinger, G., and Guy, R. K. (2003) Isotopically Labeled Crosslinking Reagents: Resolution of Mass Degeneracy in the Identification of Crosslinked Peptides. Bioorg Med Chem Lett. 13, 4023-4026; Petrotchenko, E. V., Olkhovik, V. K., and Borchers, C. H. (2005) Isotopically Coded Cleavable Cross-Linker for Studying Protein-Protein Interaction and Protein Complexes. Mol Cell Proteomics 4, 1167-1179; and Petrotchenko, E., and Borchers, C. (2010) Icc-Class: Isotopically-Coded Cleavable Crosslinking Analysis Software Suite. BMC bioinformatics 11, 64, fluorescently labeled (Sinz, A., and Wang, K. (2004) Mapping Spatial Proximities of Sulfhydryl Groups in Proteins Using a Fluorogenic Cross-Linker and Mass Spectrometry. Anal Biochem. 331, 27-32), and mass-tag labeled cross-linking reagents, for example as described in Tang, X., Munske, G. R., Siems, W. F., and Bruce, J. E. (2005) Mass Spectrometry Identifiable Cross-Linking Strategy for Studying Protein-Protein Interactions. Anal Chem 77, 311-318; and Back, J. W., Hartog, A. F., Dekker, H. L., Muijsers, A. O., de Koning, L. J., and de Jong, L. (2001) A New Crosslinker for Mass Spectrometric Analysis of the Quaternary Structure of Protein Complexes. J. Am. Soc. Mass Spectrom. 12, 222-227. These methods can identify cross-linked peptides with MS analysis, but interpretation of the data generated from inter-linked peptides (two peptides connected with the cross-link) by automated database searching remains difficult. Several bioinformatics tools have thus been developed to interpret MS/MS data and determine inter-linked peptide sequences from complex mixtures, as described in Maiolica, A. et al.; Schilling, B. et al.; Chu, F., Baker, P. R., Burlingame, A. L., and Chalkley, R. J. (2009) Finding Chimeras: A Bioinformatic Strategy for Identification of Cross-Linked Peptides. Mol Cell Proteomics 9, 25-31; Gao, Q., Xue, S., Shaffer, S. A., Doneanu, C. E., Goodlett, D. R., and Nelson, S. D. (2008) Minimize the Detection of False Positives by the Software Program Detectshift for 18o-Labeled Cross-Linked Peptide Analysis. Eur J Mass Spectrom (Chichester, Eng) 14, 275-280; Singh, P., Shaffer, S. A., Scherl, A., Holman, C., Pfuetzner, R. A., Larson Freeman, T. J., Miller, S. I., Hernandez, P., Appel, R. D., and Goodlett, D. R. (2008) Characterization of Protein Cross-Links Via Mass Spectrometry and an Open-Modification Search Strategy. Anal Chem 80, 8799-8806; Rinner, O., Seebacher, J., Walzthoeni, T., Mueller, L. N., Beck, M., Schmidt, A., Mueller, M., and Aebersold, R. (2008) Identification of Cross-Linked Peptides from Large Sequence Databases. Nat Methods 5, 315-318; Lee, Y. J., Lackner, L. L., Nunnari, J. M., and Phinney, B. S. (2007) Shotgun Cross-Linking Analysis for Studying Quaternary and Tertiary Protein Structures. J Proteome Res 6, 3908-3917; and Nadeau, O. W., Wyckoff, G. J., Paschall, J. E., Artigues, A., Sage, J., Villar, M. T., and Carlson, G. M. (2008) Crosssearch, a User-Friendly Search Engine for Detecting Chemically Cross-Linked Peptides in Conjugated Proteins. Mol Cell Proteomics 7, 739-749. Although promising, further developments are still needed to make such data analyses as robust and reliable as analyzing MS/MS data of single peptide sequences using existing database searching tools (e.g. Protein Prospector, Mascot or SEQUEST).
Various types of cleavable cross-linkers with distinct chemical properties have been developed to facilitate MS identification and characterization of cross-linked peptides. These include UV photocleavable (Nadeau, O. W., Wyckoff, G. J., Paschall, J. E., Artigues, A., Sage, J., Villar, M. T., and Carlson, G. M. (2008) Crosssearch, a User-Friendly Search Engine for Detecting Chemically Cross-Linked Peptides in Conjugated Proteins. Mol Cell Proteomics 7, 739-749), chemical cleavable (Kasper, P. T., et al.), isotopically-coded cleavable (Petrotchenko, E. V., et al.), and MS-cleavable reagents, as described in Tang, X, et. al.; Back, J. W., et. al.; Zhang, H., Tang, X., Munske, G. R., Tolic, N., Anderson, G. A., and Bruce, J. E. (2009) Identification of Protein-Protein Interactions and Topologies in Living Cells with Chemical Cross-Linking and Mass Spectrometry. Mol Cell Proteomics 8, 409-420; Soderblom, E. J., and Goshe, M. B. (2006) Collision-Induced Dissociative Chemical Cross-Linking Reagents and Methodology: Applications to Protein Structural Characterization Using Tandem Mass Spectrometry Analysis. Anal Chem 78, 8059-8068; Soderblom, E. J., Bobay, B. G., Cavanagh, J., and Goshe, M. B. (2007) Tandem Mass Spectrometry Acquisition Approaches to Enhance Identification of Protein-Protein Interactions Using Low-Energy Collision-Induced Dissociative Chemical Crosslinking Reagents. Rapid Commun Mass Spectrom 21, 3395-3408; Lu, Y., Tanasova, M., Borhan, B., and Reid, G. E. (2008) Ionic Reagent for Controlling the Gas-Phase Fragmentation Reactions of Cross-Linked Peptides. Anal Chem 80, 9279-9287; and Gardner, M. W., Vasicek, L. A., Shabbir, S., Anslyn, E. V., and Brodbelt, J. S. (2008) Chromogenic Cross-Linker for the Characterization of Protein Structure by Infrared Multiphoton Dissociation Mass Spectrometry. Anal Chem 80, 4807-4819. MS-cleavable cross-linkers have received considerable attention since the resulting cross-linked products can be identified based on their characteristic fragmentation behavior observed during MS analysis. Gas-phase cleavage sites result in the detection of a “reporter” ion (Back, J. W., et al.), single peptide chain fragment ions (Soderblom, E. J., and Goshe; Soderblom, E. J., Bobay, B. G., et al.; Lu, Y., et al. and Gardner, M. W. et al.), or both reporter and fragment ions (Tang, X., et al.; and Zhang, H. et. al.). In each case, further structural characterization of the peptide product ions generated during the cleavage reaction can be accomplished by subsequent MSn1 analysis. Among these linkers, the “fixed charge” sulfonium ion containing cross-linker developed by Lu. et. al appears to be the most attractive as it allows specific and selective fragmentation of cross-linked peptides regardless of their charge and amino acid composition based on their studies with model peptides.
Despite the availability of multiple types of cleavable cross-linkers, most of the applications have been limited to the study of model peptides and single proteins. Additionally, complicated synthesis and fragmentation patterns have impeded most of the known MS-cleavable cross-linkers from wide adaptation by the community.